The Cancer Modeling Section seeks to elucidate the complex molecular/genetic program governing tumor genesis and progression through the development and analysis of genetically engineered mouse models of human cancer. Our efforts in this regard are focused primarily on cutaneous malignant melanoma. Exposure to ultraviolet radiation (UV) is a causal agent in the vast majority of melanoma. Previously, we tested this hypothesis in transgenic mice in which the receptor tyrosine kinase MET was deregulated by virtue of ectopic expression of its ligand, hepatocyte growth factor/scatter factor (HGF). We discovered that a single neonatal dose of burning UV in these mice was necessary and sufficient to induce tumors reminiscent of human melanoma with shortened latency (Noonan et al., Nature 413: 271-2, 2001). A critical role for the INK4A/ARF locus, which helps regulate the pRb and p53 pathways and is widely regarded as a key melanoma suppressor in human patients, was also confirmed in our animal model (Recio et al., Cancer Res. 62: 6724-30, 2002). There has been controversy surrounding the relative risks associated with UVB versus UVA radiation. We used albino HGF transgenic mouse to show that UVB, but not UVA, alone is able to induce the full melanoma phenotype in the absence of pigment (DeFabo et al., Cancer Res. 64: 6372-6, 2004). However, we more recently showed that UVA is highly melanomagenic in pigmented HGF/SF-transgenic mice (Noonan et al., Nat. Commun. 3: 884, 2012), demonstrating that melanin is associated with oxidative DNA damage and mutagenesis, and thus represents a double-edged sword with respect to melanoma risk. Our work suggests that indoor tanning, which is mostly UVA-based, could be a significant health risk. We have also employed in vitro and in vivo models based on genetically engineered melanocytes to identify novel regulators of differentiation, malignancy and metastasis. For example, we compared the oncogenic roles of the three major NRAS downstream effectors, RAF, PI3K and RAL guanine exchange factor (RalGEF) (Mishra et al., Oncogene 29:2249-2256, 2010). Although no single downstream pathway could recapitulate all the consequences of oncogenic NRAS, we discovered that constitutive RalGEF activation stimulated anchorage-independent growth, indicating that this often overlooked pathway should be evaluated as a possible therapeutic target. We have, in collaboration with Jim McMahon and Curt Henrich (Molecular Targets Development Program) also begun re-exploring the prospects of differentiation therapy. INK4A/ARF-deficient melanocytes transformed by mutant NRAS were used in a high-throughput screen of the LOPAC drug library to identify agents that would induce re-differentiation of malignant melanoma cells, thus converting them to a more benign state. Eight candidate drugs were identified that induce a differentiated morphology. Interestingly, several of these candidates were related to dopaminergic signaling, including inhibitors of DOPA decarboxylase (DDC), which we found to be upregulated in melanomas. Dopamine not only controls the biosynthesis and secretion of melanotropins, but also helps regulate the activity of cAMP, which is also linked to melanin production. The differentiating activity of these compounds was confirmed in mouse and human melanoma cells. Notably, we found that several of these compounds effectively reduced the ability of melanoma cells to invade and grow in soft agar as well. The data suggest that re-differentiating drugs can also have anti-tumorigenic properties. Although an extensive accumulation of epidemiological evidence supports a fundamental role for UV in melanoma, the specific UV-affected molecular pathways and mechanisms remain largely unidentified. We have suggested that mechanisms other than UV-induced DNA mutagenesis may also be important in melanoma initiation. To determine the role(s) of UV in melanoma in vivo, we developed a mouse model (iDCT-GFP) that allowsmelanocytes, specifically and inducibly labeled with green fluorescent protein (GFP), to be highly purified from disaggregated mouse skin by FACS following UV irradiation in vivo. We identified a pattern of UVB induced gene expression changes in melanocytes isolated from mice that are consistent with inflammatory alterations and may spare melanocytes post-UV remodeling-associated destruction. We have identified an interferon (IFN)-gamma signaling signature arising in melanocytes after neonatal UV irradiation. The source was a type of macrophage recruited to the skin after UV exposure; IFN-gamma in turn activated melanocytes and the expression of genes that could facilitate immunoevasion. Transplanted neonatal macrophages were found to significantly enhance melanoma growth in vivo in an IFN-gamma-dependent fashion. This was surprising considering that IFN-alpha has been used to treat melanoma patients, albeit with limited success. We hypothesized that melanomas escape immune destruction by co-opting these pathways already hard-wired in melanocytes, and suggested that the IFN-gamma signaling pathway may represent a promising therapeutic target for melanoma patients (Zaidi et al., Nature 469:548-553, 2011). To address the UV mutation question we are also subjecting in vivo-exposed melanocytes to whole exome and RNA sequencing. We are isolating GFP-labeled melanocytes from all stages of melanoma development relevant to human disease in an attempt to catalog their precise genomic alterations. We anticipate that this in vivo model will provide novel insights into the nature of UV-induced damage, and the mechanisms by which UV provokes melanoma. We have also hypothesized that late stage melanoma cells can co-opt pathways hard-wired into normal developing melanoblasts to achieve a more aggressive and metastatic phenotype. Both the embryonic melanoblast and the metastatic melanoma cell must undergo a similar EMT and become invasive, highly migratory, and survive to colonize at a remote sites. We again employed our iDCT-GFP model to isolate embryonic melanoblasts from key stages of melanocyte development. RNA sequencing and microarray-based gene expression profiling have been performed from representative developmental stages. Genes have been identified whose expression is characteristically up-regulated (or down-regulated) in both melanoblasts and metastatic melanoma relative to adult melanocytes, which may represent new therapeutic targets against metastasis in melanoma. Novel candidates, which include genes that regulate ER stress, autophagy, and neural developoment have been evaluated for a role in metastasis using siRNA knockdown in metastatic human and mouse melanoma cells and tail vein injections. Several candidates have now been shown to regulate metastatic behavior, and to correlate with melanoma patient survival. These are being vigorously pursued. Finally, we integrated gene and microRNA expression data from our mouse models of highly and poorly malignant melanocytic tumors, and human melanoma databases, and discovered an important role for pathways centered on a new tumor suppressor, miR-32. Malignant tumors frequently exhibited poor expression of miR-32, the targets for which included NRAS, PI3K and notably, myeloid cell leukemia 1 (MCL1). MCL1 was highly expressed in melanomas, and when knocked down diminished oncogenic potential. MCL1 overexpression transformed immortalized primary mouse melanocytes, but only those expressing activating mutations in BRAF, CRAF or PI3K. The MCL1-specific antagonist sabutoclax was effective as a single agent, and acted synergistically in combination with vemurafenib in preclinical melanoma models. Our data showed that miR-32/MCL1 pathway members are novel candidate anti-melanoma drug targets, and suggested that their inhibition may enhance the efficacy of BRAFV600E inhibitors in the clinic.